Evaluation of some Haematological and Immunologic Parameters as Adjunct marker to Viral Load in HBV Management in a Resource-Constrained Setting

doi:10.21203/rs.3.rs-2377152/v1

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Evaluation of some Haematological and Immunologic Parameters as Adjunct marker to Viral Load in HBV Management in a Resource-Constrained Setting

https://doi.org/10.21203/rs.3.rs-2377152/v1

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published 06 Apr, 2023

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Introduction: In a resource-constrained situation where HBV DNA viral load testing may not be available or be prohibitively expensive, the current study sought to assess the levels of white blood cells, red blood cells, haemoglobin, platelets count and other red cell indices, CD4+ T-cell count and ALT as an adjunct marker to viral load in HBV Management.

Methods: A case-control cross-sectional study was conducted among 300 subjects (100 acute HBV patients, 100 chronic HBV patients, and 100 HBV-negative individuals). The haemostatic parameter, absolute CD4+ T-cell count, HBcAb-IgM, alanine aminotransferase (ALT) and HBV-DNA were measured using standard methods.

Results: When compared to HBV-negative individuals, the median (interquartile range, IQR) of acute and chronic HBV-infected participants had significantly higher total WBC, granulocyte, and monocyte counts, but lower platelet, lymphocyte, and RBC counts. When compared to the HBV-negative group, the median (IQR) of acute and chronic HBV-infected participants had significantly lower haemoglobin, haematocrit, mean cell volume, mean cell haemoglobin, and mean cell haemoglobin concentration. ALT levels were highest in acute HBV participants (median 61.0 [IQR: 48.75-72.00]) and lowest in HBV negative controls (median 24.50 [IQR: 20.0-36.75]) (Krustal-Wallis test= 151.2, p <0.0001). HBV negative controls had the highest CD4+ T-cell count levels (median 784.0 [IQR: 548.0-953.8] and lowest in acute HBV infected participants (median 194.5 [IQR: 142.0-308.0]) (Krustal-Wallis test= 193.2, p < 0.0001)

Conclusion: Our findings highlight the impact of HBV infection on iron metabolism and the production of haematological precursor cells. As such these parameters could be used as adjunct markers for viral load in the management of HBV infection in a resource-constrained setting.

CD4+ T-cell count

HBV-DNA

Haematological

immunological parameters

ALT

Hepatitis B virus (HBV) is an important hepatotropic DNA virus which presents with a variety of pathological conditions and causes acute or chronic liver infection. The majority of infected adults can resolve the infection, but 5–10% develop chronic hepatitis, which can lead to cirrhosis, hepatocellular carcinoma (HCC) and death [1, 2]. The virus can be transmitted parenterally, perinatally and sexually, and infection can be prevented by infant vaccination or reducing exposure to particular lifestyles and environmental risk factors [1], [3]. The acute form of hepatitis B is associated with the sero-presence of the HBcAb-IgM type and the presence of HBsAg and HBeAg. Whereas, the sero-presence of HBcAb-IgG type as well as HBsAg for more than six months is considered indicative of chronic HBV infection [4]. However, one of the frequently identified extrahepatic abnormalities often seen at the time of diagnosis of acute viral hepatitis is the haematological abnormality [5].

Measurement of HBV DNA viral load may not be available or may be too expensive to deploy in the management of HBV infection in a resource-constrained setting. Haematological parameters such as red blood cells (RBC), white blood cells (WBC), and platelets are blood cells in the complete blood count that have unique functions [6]. Studies have shown that these cells have altered values in disease conditions such as viral hepatitis [7]. Alanine aminotransferase (ALT) is among the enzymes expressed following cell death or injury. It serves as a biomarker, and its levels in serum could assist in the diagnosis and prognosis of diseases and injuries caused by viral hepatitis to the liver [7]. In acute HBV infection, there is usually an increase in aminotransferases, especially the ALT level, from a moderate to an astronomical increase of more than 100 folds. Chronic HBV infection however usually presents with normal or elevated ALT levels of up to 200 IU/L in about 90% of patients [3, 8, 9].

Therefore, the varying severity of hepatitis and other liver diseases associated with various stages of HBV infection, including the acute and chronic stages, may also produce varying degree of abnormal haematological parameters. Hence, the current study aimed to investigate the levels of white blood cells, red blood cells, haemoglobin, platelets count and other red cell indices, CD4 + T-cell count and ALT as an adjunct marker to HBV-DNA in the Management of Acute and Chronic HBV Infection in a resource-constrained setting.

Study Setting and Design

This case-control cross-sectional study assessed some haematological and immunological parameters in HBV-negatives as control and compared them with acute and chronic HBV patients. This study was conducted between April 2021 and July, 2022 at the antiretroviral therapy (ART) Laboratory of the Ahmadu Bello University Teaching Hospital (ABUTH), Zaria, Nigeria.

Study Participants

A total of three hundred (300) adults aged 18-80 years who were chosen by convenience sampling participated in this study. The breakdown of the study participants includes one hundred (100) confirmed HBV-negative individuals as controls, one hundred (100) confirmed acute HBV-infected and one hundred (100) chronic HBV-infected patients.

Sociodemographic and clinical data

Relevant sociodemographic characteristics and clinical data were obtained using a structured questionnaire and a review of the patient’s clinical records.

Specimens Collection and Laboratory Analyses

Ten millilitres (10 mL) of venous blood were collected from each participant into EDTA and plain bottles using aseptic techniques. All blood samples collected in the EDTA tubes were immediately analyzed for Haematological profile, CD4⁺cell count and HBV-DNA viral load. Samples for HBV-DNA assays that could not be processed immediately were stored at -80⁰C until analysis. The sera harvested from the plain tubes were used for serology and ALT Assay.

Detection of HBsAg by ELISA

The surface antigen for hepatitis B was tested by the ELISA technique on all specimens using the method of Burtis et al. [10]. Fortress Diagnostics’ 4th generation ELISA kit (Ireland, UK) was used. The manufacturer’s instructions on testing were strictly followed.

HBcAb-total and HBcAb-IgM test

HBV-infected (HBsAg positive) subjects were tested for HBcAb-total, while those positive for HBcAb-total were further tested for HBcAb-IgM, which is a marker of acute HBV infection. Subjects who tested positive for HBcAb-IgM were classified as having an acute infection. However, subjects who tested negative for HBcAb-IgM were classified as having a chronic infection. The Advance Quality ^TMOne Step HBcAb-total and HBcAb-IgM test kits (InTec Products China) were used for these tests.

Estimation of Absolute CD4⁺ T Cells Count

The CD4⁺ T-cell count estimation was performed using the Cyflow counter machine (Partec, Germany) that uses the principle of flow cytometry [11]. All testing protocols were conducted based on the manufacturer’s instructions.

Estimation of Full Blood Count, Red Cell Indices and Platelet Count

Full blood counts of all participants in this study were carried out using the Mindray BC – 5000 5-part differential Auto haematology analyzer. The measurement methods used in this analyzer are; the Electrical Impedance method for determining the Red Blood Cell (RBC) and Platelet (PLT) data, the colorimetric method for determining the haemoglobin, flow Cytometry by laser for determining the white blood cell data. Other parameter results including the Red Blood Cell indices (MCV, MCH, MCHC) were obtained through automated calculation.

Determination of Alanine Amino Transferase (ALT)

Alanine Aminotransferase was determined by a standard method as described by Reitman and Frankel [12] using Randox reagents and Stax Fax 1904 Spectrophotometer. Alanine Aminotransferase (ALT) determination was measured by monitoring the concentration of pyruvate hydrazone formed with 2, 4- dinitrophenylhydrazine. The kit manufacturer instructions and procedures were strictly followed.

HBV-DNA plasma RNA Isolation, Amplification and Quantification

HBV viral load was performed using the COBAS^®Ampliprep/ COBAS^® Taqman^® HBV DNA Test, version 2 (Roche Diagnostics, USA). This automated amplification and quantification have a detection limit of between 20–170,000,000 IU/mL. Sample preparations and polymerase chain reaction (PCR) amplification were performed according to the method described by Osuji et al. [9]. The Standard Operating procedures were strictly followed to ensure accurate laboratory findings following the manufacturer’s instructions.

Statistical analyses

Data obtained were presented in tables as percentages and median (interquartile range [IQR] after failing the normality check. Comparison among groups was analyzed using analysis of variance (ANOVA) while comparison between groups was done using post Hoc analysis. Pearson’s correlation was used to determine the relationship and association between parameters using Graphpad prism software (v 6) respectively. Significance level was set at P ≤0.05 and 95% confidence interval

Table 1 shows the demographic analysis of the study participants. Overall, 300 participants were recruited in the study, which comprises 100 HBV-negative control subjects, 100 Acute HBV infected participants and 100 Chronic HBV infected participants. The age of the studied participants ranged from 18 to 80 with their mean ages (39.55±13.48, 38.7±14.94 and 38.85±14.92 years) respectively. The sex of HBV-negative control participants, Acute HBV and Chronic HBV-infected participants were as follows: 32% females and 68% males; 44% females and 56% males; 25% females and 56% males (Table 1).

Comparison of Total White Blood Cells, Red Blood Cells, Platelet, Lymphocyte and Granulocyte counts in the study participants

The median (interquartile range, IQR) of acute and chronic HBV-infected participants recorded significantly high total WBC and Granulocyte counts; and low platelet, lymphocyte and RBC counts, when compared with the HBV negative control (Table 2).

Comparison of haemoglobin, haematocrit, mean cell volume, mean cell haemoglobin, and mean cell haemoglobin concentration in the study participants

The median (interquartile range, IQR) of acute and chronic HBV-infected participants recorded significantly low haemoglobin, haematocrit, mean cell volume, mean cell haemoglobin, and mean cell haemoglobin concentration, when compared with the HBV-negative control. (Table 3)

Alanine transaminase (ALT) levels of Acute HBV(A), Chronic HBV(C) and HBV negative control (H) in the study population

Figure 1 compares the median (IQR) of ALT levels of HBV negative control (H), Acute HBV(A) and Chronic HBV(C) in the study population. ALT levels were highest in Acute HBV participants (median 61.0 [IQR: 48.75–72.00]), followed by chronic HBV participants (median 44.00 [IQR: 30.25–59.50]), and lowest in HBV negative control (median 24.50 [IQR: 20.0–36.75]) (Krustal-Wallis test = 151.2, p < 0.0001)

CD4+ T-cell count levels among HBV negative control (H), Acute HBV(A) and Chronic HBV(C) in the population

The CD4+ T-cell count levels were highest in HBV negative control (median 784.0 [IQR: 548.0-953.8), followed by Chronic HBV infected participants (median 198 [IQR: 172.5.0-218.0]), and lowest in acute HBV infected participants (median 194.5 [IQR: 142.0-308.0]) (Krustal-Wallis test = 193.2, p < 0.0001) Figure 2).

Comparison of HBV DNA of Acute HBV(A) and Chronic HBV(C) infection in the study population

The HBV-DNA levels were highest in Chronic HBV infected participants (median 2.96 [IQR: 2.42- 5.96; 95% CI- 3.602 to 4.532) and lowest in acute HBV-infected participants (median 2.50 [IQR: 1.70-2.74]; 95% CI-2.266 to 2.741) (p < 0.0001, Figure 3).

Despite the implementation of vaccination programmes, hepatitis B virus (HBV) infections remain a global health problem [2]. Thus, haematological parameters have a unique function and studies have shown that these cells’ functions and values are altered in disease conditions such as viral hepatitis [7]. In this study, we assessed some haematological and immunological parameters as adjunct markers to viral load in the Management of Acute and Chronic HBV Infection in a resource-constrained setting.

The median (interquartile range, IQR) of acute and chronic HBV-infected participants recorded significantly high total WBC, granulocyte and monocyte counts; and low platelet, lymphocyte and RBC counts, when compared with the HBV negative control. These abnormal haematological parameters could be due to response to tissue injury, and inflammation [13, 14, 15]. Several researchers have reported that haematological parameters provide valuable information as regards the manifestation of HBV infection since the condition may lead to changes in haemostatic values such as platelet, Packed Cell Volume (PCV), Haemoglobin (Hb) and White Blood Cell (WBC) disorders which include; absolute changes in Leukocyte numbers, involving Neutrophil, Lymphocyte and Eosinophil in response to viral infection and inflammation [15, 16]. It is possible that inflammation caused by HBV infection has an effect on the production of haematological precursor cells from a pluripotent stem cell. Research has shown significantly raised absolute leukocytes, neutrophils, lymphocytes and eosinophils as well as monocytosis in HBV infection [15, 17, 18].

The levels of haemoglobin, PCV, MCV, MCH and MCHC were significantly lower in the HBV acute, chronic infection when compared with the control group (P < .05). This could be attributed to temporary bone marrow suppression and autoimmune haemolytic anaemia which may accompany viral hepatitis. Studies had shown that some abnormal haematological parameters in HBV infection include defects in levels of platelet numbers, packed cell volume (PCV), haemoglobin (Hb) and white blood cell (WBC) disorders which include absolute changes in Leukocyte numbers, involving neutrophils lymphocytes and eosinophils in response to tissue injury, and inflammation [13, 14, 15]. Packed cell volume in most patients with acute viral hepatitis gradually decreases during the first three weeks of illness [5]. It has been well established that many haematological abnormalities occur in HBV infection possibly due to cell distortions that occur following inflammation caused by the infection which may likely result in alterations in iron metabolism, aberrant production of haematological precursor cells as well as a defect in red blood cell morphology [15, 16].

In this study, ALT levels were found to be highest in Acute HBV patients and chronic HBV patients when compared to HBV negative control. These abnormal haematological parameters recorded by both the acute and chronic subjects could also be attributed to the indirect consequences of hepatic damage (hepatitis) of both subjects, evident in their abnormally high level of ALT values, and directly to the ability of HBV to inhibit haematopoiesis among these patients [19]. Outside its role as an extravascular haemotopoietic organ in early foetal life and bone marrow infiltrative disease, the liver synthesizes and stores many of the elements and proteins necessary in blood production. It also plays a crucial role in haemostasis [5].

The study observed low values of CD4 + T-cell count among acute and chronic HBV participants when compared to HBV negative control. This could be due to acute and chronic infection [20]. It has been shown that low CD4 T-cell count could be due to immunosuppression and the primary target for viral infection is CD + T-lymphocyte [21]. Trautmann et al. [22] showed that the depletion of CD4 + T cells can lead to chronic derailed liver function.

This study found abnormal high values of total WBC, granulocyte, ALT and low platelet, lymphocyte and RBC counts in acute and chronic HBV patients. This study also found decreased values of some blood cells in acute and chronic HBV patients. HBV infection has an effect on iron metabolism and production of haematological precursor cells which are reflected in low levels of haemoglobin, PCV, MCV, MCH and MCHC as such these parameters could be used as an adjunct marker to augment HBV DNA viral load testing in managing HBV infection in Nigeria.

Funding

No funding was received

Conflicts of interest/Competing interests

The authors declare that they have no conflict of interests

Ethics approval

The study protocol was approved by the hospital research ethics committee of ABUTH, Zaria, Nigeria (ethical approval number: ABUTHZ/HREC/B23/2019). Informed consent was obtained from all the participants in accordance with Helsinki Declaration of 1975, as revised in 2000; voluntariness and strict confidentiality were maintained throughout the study.

Consent to participate

Written consent was taken from all the participants.

Consent for publication

Not applicable

Availability of data and material

The data sets used and/or analyzed during the current study are available from the corresponding author on request.

Code availability

Not applicable

Authors Contributions

BIS conceived the study. BIS, MM, BTB and OA carried out the laboratory assay. BIS and AU performed the statistical analysis. BIS, AU MAH, AD, MMA, OOP, AD, LL and SSI participated in the design of the study and help to draft the manuscript. BIS, AU, MM, MMA, OOP and MAA revised the manuscript and coordinated the study. All authors read and approved the final manuscript

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Table 1: Age and Sex Distribution of the study Participants

Demographics

H=HBV negative control (N=100)

A=Acute HBV infection (N=100)

C=Chronic HBV infection (N=100)

Age (years), Mean±SD

39.55±13.48

38.7±14.94

38.85±14.92

Gender, n (%)

Female

Male

Table 2: Comparison of Total White Blood Cells, Red Blood Cells, Platelet, Lymphocyte, Granulocyte and Monocyte counts in the study participants

Participants	Median (IQR)
Participants	WBC (x10⁹/L)	RBC (x10⁹/L)	PLT (x10⁹/L)	LYMP (x10⁹/L)	GRAN (x10⁹/L)	MONO (x10⁹/L)
H=100	4.6(3.8-5.7)	3.9(3.7-4.3)	252(216-290)	4.2(2.8-5.3)	2.6(1.9-3.4)	0.6(0.40-0.80)
A=100	5.6(4.3-9.5)	2.6(2.2-2.9)	215(159-298)	1.6(0.7-3.1)	3.6(2.9-4.5)	0.8(0.60-1.50)
C=100	5.5(4.4-7.7)	2.5(2.1-2.9)	214(160-296)	1.5(0.6-3.1)	2.9(2.1-4.5)	0.9(0.60-1.50)
Krustal-Wallis	20.78	160.3	10.81	82.27	40.25	32.01
P-value	<0.0001	<0.0001	0.0045	<0.0001	<0.0001	<0.0001
H vs. A	<0.0001	<0.0001	0.0155	<0.0001	<0.0001	<0.0001
H vs. C	0.0007	<0.0001	0.0115	<0.0001	0.0204	<0.0001
A vs. C	>0.9999	>0.9999	>0.9999	>0.9999	0.0009	>0.9999

H=HBV negative control

A=Acute HBV infection

C=Chronic HBV infection

Table 3: Comparison of haemoglobin, haematocrit, mean cell volume, mean cell haemoglobin, and mean cell haemoglobin concentration in the study population

Participants	Median (IQR)
Participants	HCT (%)	HGB (g/dL)	MCV(fl)	MCH(Pg)	MCHC (g/L)
H=100	40.1(36.6-45.6)	13.1(12.4-14.6)	85.2(82.7-88.8)	28.4(27.3-29.73)	32.8(31.5-34.3)
A=100	33.8(30.5-37.2)	11.1(10.1-12.5)	74.7(73.1-78.0)	27.9(25.6-29.7)	32.2(30.7-33.1)
C=100	33.8(30.5-36.8)	11.1(10.1-12.1)	74.7(72.9-78.0)	27.9(25.6-29.7)	31.9(30.7-33.10)
Krustal-Wallis	94.23	98.97	2.028	6.515	7.015
P-value	<0.0001	<0.0001	0.3627	0.0385	0.0300
H vs. A	<0.0001	<0.0001	0.7718	0.1004	0.0838
H vs. C	<0.0001	<0.0001	0.5670	0.0670	0.0523
A vs. C	>0.9999	>0.9999	>0.9999	>0.9999	>0.9999

H=HBV negative control

A=Acute HBV infection

C=Chronic HBV infection

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Journal Publication

published 06 Apr, 2023

Read the published version in SN Comprehensive Clinical Medicine →

Reviewers agreed at journal
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Reviewers invited by journal
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Editor assigned by journal
10 Jan, 2023
First submitted to journal
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Evaluation of some Haematological and Immunologic Parameters as Adjunct marker to Viral Load in HBV Management in a Resource-Constrained Setting

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